Therapeutic manipulation and spatial quantification of the tumor microenvironment in colorectal cancer.

Colorectal cancer (CRC) is a complex ecosystem shaped by bidirectional interactions between epithelium and the tumor microenvironment, prominently mediated by TGFβ signaling. Cancer-associated fibroblasts (CAFs) are regulators of epithelial plasticity and immune cell recruitment; yet, their diversity has impacted translationally applicable spatial analysis. Here, we distil the fibroblast continuum into two overarching CAF populations that are largely transcriptomically distinct and are marked by PDGFRA(+) and ACTA2(+) expression, enabling robust spatial identification using single immunohistochemical markers. We show that TGFβ signaling drives dynamic transitions between these states. In a preclinical model, selective ALK5 inhibition remodels CAF composition in vivo, reconfiguring local immune neighborhoods and indirectly altering epithelial stem cell states. Finally, we demonstrate that multiscale spatial analysis provides a quantitative readout of stromal-immune-epithelial remodeling following therapy. These findings establish a simplified, translationally relevant CAF framework and highlight spatially resolved stromal dynamics as measurable indicators of therapeutic response in CRC.